How transmembrane (TM) domains of membrane proteins transmit the signal across the cell membrane has long been a subject of keen interest in biology. There is a recent paradigm shift in the mechanism of activation for the cytokine receptor superfamily. The role of cytokine hormone binding to the extracellular domain is now recognized as an inducer of the conformational change of pre-dimerized TM domains that triggers subsequent intracellular responses. This is drastically different from its traditional role as an organizer whose sole function was to initiate the receptor TM dimer formation. Our long-term objective is to delineate the mechanisms and accompanying energetics of TM-induced signaling of various single-pass TM receptors during the inactive to active transition upon ligand binding. Our hypothesis is that the inactive off-state conformation is much more stable than the active on-state one, and the major role of ligand binding is to disrupt the pre-dimerized (energetically stable) TM-TM contact that locks-in the off-state structure, to direct the (energetically unstable) on-state structure. In this proposal, we will use human growth hormone receptor (hGHR) and human prolactin receptor (hPRLR) as prototypical model systems for homodimeric TM-induced activation. The objectives of this proposal are to determine the interfacial residues of hGHR and hPRLR TM dimers and to elucidate the conformational and energetic changes during the activation process by innovative, multidisciplinary combination of versatile computational and experimental approaches. The successful completion of this project will have a significant impact on the field, not only by elucidating the TM signaling mechanism and energetics, but also by providing the computational and experimental methods that can be used to characterize the biological activation process of other cytokine receptors and the plentitude of other single-pass TM receptors, which all have the critical importance to biology and thus, human health.